Method of separating pancreatic islet

ABSTRACT

The present invention provides a pancreatic islet isolation method comprising the steps of (1) injecting a protection solution containing a protease inhibitor into the pancreatic duct of an procured pancreas; (3) digesting the pancreas into which the protection solution has been injected; and (4) purifying the digested pancreatic tissue using a purification solution containing a density gradient reagent. The present invention also provides a protection solution for injection into the pancreatic duct, a pancreas preservation solution for the two-layer method, and an islet purification solution.

TECHNICAL FIELD

The present invention relates to a method of isolating pancreaticislets, which is a critical technology for pancreatic islettransplantation. More particularly, the present invention relates to aprotection solution for injection into the pancreatic duct, a pancreaspreservation solution for the two-layer method, and an isletpurification solution, each of which is well-adapted for use inpancreatic islet isolation.

BACKGROUND ART

The technique of pancreatic islet transplantation into type-1 diabeticpatients, who are unable to survive without the administration ofinsulin, that is, who are in an insulin dependent diabetes mellitus, isgarnering a great deal of public awareness and efforts are being made,mainly in Europe and the United States, to establish this technique as aclinical treatment.

Pancreatic islet transplantation refers to cellular tissuetransplantation in which pancreatic islet cell groups, which play acentral role in blood sugar regulation in the body, are administered byinfusion into the portal vein. Islet transplantation is minimallyinvasive for the transplant recipient and is regarded as the treatmentnearest to ideal for type-1 diabetic patients.

In 2000, at the University of Alberta in Edmonton, Canada, a successfultrial of clinical islet transplantation was reported. Since this report,approximately 300 islet transplantations have been performed in the 4years, mainly in Europe and the United States. These islettransplantations have been carried out on the basis of the Edmontonprotocol established at the University of Alberta.

However, with the technology heretofore, consistent islet yields havenot been obtained, even in islet transplantation from brain-dead donorscarried out in Europe and the United States, and in some instances thetransplanted islets have also not functioned effectively. Moreover, evenwhen considered on a worldwide basis, there have been almost nosuccessful cases of islet transplantation from non-heart-beating donors,where the conditions are worse than with brain-dead donors, and in factislet transplantation from non-heart-beating donors has to date not beenpossible.

To raise the success rates of islet transplantation and also to achievesuccessful islet transplantation from non-heart-beating donors, it isimportant to transplant a large population of islets fit fortransplantation. Therefore, there has been strong demand forimprovements in islet isolation technology in order to raise the yieldof transplantable islets.

On the other hand, in the medical treatment of transplantation, a methodhas been reported in which ulinastatin or a ulinastatin substitute isadministered post-transplant to organ transplant patients (See JapaneseUnexamined Patent Publication No. 2002-20309).

Further, a solution for perfusion or storage of organs that are destinedfor transplantation has been reported, wherewith excellent results wereobtained in lung transplantation (refer to Japanese Unexamined PatentPublication No. H6-40801).

However, an optimal means for islet transplantation, particularly withregard to islet isolation and purification technology, remains elusive.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

A main object of the present invention is to provide islet isolationtechniques that can improve the yield of transplantable islets.

Means for Solving the Problems

The present inventors conducted varied and extensive investigations withthe main goal of improving islet yields, and as a result, they foundthat the yield of transplantable islets is raised by the use of specialsolutions and methods. They conducted further intensive research andachieved the present invention.

That is, the present invention relates to the following isolationmethods and solutions.

Item 1: A pancreatic islet isolation method comprising the steps of:

(1) injecting a protection solution containing a protease inhibitor intothe pancreatic duct of an procured pancreas;

(3) injecting an enzyme solution into said pancreas into which theprotective solution has been injected and digesting the pancreas; and

(4) purifying the digested pancreas tissue using a purification solutioncontaining a density gradient reagent.

Item 2: An isolation method according to Item 1, wherein the protectionsolution is injected into the pancreatic duct at the rate ofapproximately 0.1 to 10 ml per 1 gram organ weight in step (1).

Item 3: An isolation method according to Item 1 or 2, wherein theprotease-inhibitor-containing protection solution has a potassiumconcentration of approximately 4 to 50 mmol/L.

Item 3A: An isolation method according to any of Items 1 to 3, whereinthe protease-inhibitor-containing protection solution further containstrehalose.

Item 4: An isolation method according to any of Items 1 to 3A, furthercomprising (2) a step of preserving the protection-solution-injectedpancreas by a two-layer method.

In other words, a pancreatic islet isolation method, comprising thesteps of:

(1) injecting a protection solution containing a protease inhibitor intothe pancreatic duct of an procured pancreas;

(2) preserving by the two-layer method the pancreas into which theprotection solution has been injected;

(3) introducing an enzyme solution into the pancreas after preservationand digesting the pancreas; and

(4) purifying the digested pancreas tissue using a purification solutioncontaining a density gradient reagent.

Item 5: An isolation method according to Item 4, wherein the two-layermethod of step (2) is a two-layer method that uses (i) a liquidperfluorocarbon and (ii) a preservation solution that contains aprotease inhibitor and has a potassium concentration of approximately 4to 50 mmol/L.

Item 5A: An isolation method according to Item 5, wherein thepreservation solution (ii) further contains trehalose.

Item 6: An isolation method according to any of Items 1 to 5A, whereinthe purification solution in step (4) further contains trehalose.

Item 7: An isolation method according to any of items 1 to 6, whereinthe density gradient reagent in step (4) is iodixanol.

Item 8: An isolation method according to any of Items 1 to 7,

wherein the purification solution in step (4) further contains aprotease inhibitor.

Item 8A: An isolation method according to any of Items 1 to 8, whereinthe protease inhibitor is at least one selected from the groupconsisting of ulinastatin, gabexate mesilate, and nafamostat mesilate.

Item 9: A protection solution for injection into the pancreatic ductthat contains a protease inhibitor.

In an alternative formulation, a use of a protease-inhibitor-containingprotection solution is for injection into the pancreatic duct duringislet isolation. The protease inhibitor is preferably at least oneselected from the group consisting of ulinastatin, gabexate mesilate,and nafamostat mesilate.

Item 10: A protection solution according to Item 9 that has a potassiumconcentration of approximately 4 to 50 mmol/L.

Item 10A: A protection solution according to Item 9 or 10 that furthercontains trehalose.

Item 11: A pancreas preservation solution for a two-layer method thatcontains a protease inhibitor and has a potassium concentration ofapproximately 4 to 50 mmol/L.

In other words, a use of a preservation solution containing a proteaseinhibitor and having a potassium concentration of approximately 4 to 50mmol/L for pancreas preservation by the two-layer method during isletisolation.

The protease inhibitor is preferably at least one selected from thegroup consisting of ulinastatin, gabexate mesilate, and nafamostatmesilate.

Item 11A: A preservation solution according to Item 11 that furthercontains trehalose.

Item 12: A pancreatic islet purification solution that contains adensity gradient reagent and trehalose.

In other words, a use of a solution containing trehalose and a densitygradient reagent for islet purification during islet isolation.

Item 13: A purification solution according to Item 12, wherein thedensity gradient reagent is iodixanol.

In other words, a purification solution that contains iodixanol andtrehalose.

Item 14: The purification solution according to Item 12 or 13 thatfurther contains a protease inhibitor.

In other words, the purification solution according to Item 12 or 13,that contains a density gradient reagent, trehalose, and a proteaseinhibitor.

The protease inhibitor is preferably at least one selected from thegroup of ulinastatin, gabexate mesilate, and nafamostat mesilate.

The present invention also encompasses the following modes.

Item 15: A method of pancreatic islet isolation comprising the steps of:

(1) injecting a tissue protection or preservation solution that containsa trypsin inhibitor into the pancreatic duct of an procured pancreas;

(2) distending the pancreas by injecting a collagenase solution into theaforesaid pancreas into which the protection or preservation solutionhas been injected;

(3) activating the collagenase by raising the temperature of thesolution within the distended pancreas and thereby digesting thepancreatic tissue;

(4) recovering the digested pancreatic tissue; and

(5) purifying the islets by isolating the islets from the recoveredpancreatic tissue.

Item 16: An isolation method according to Item 15, wherein step (5)comprises the following steps (5-1) to (5-3) of:

(5-1) setting up a density gradient in a purification solutioncomprising a tissue protection or preservation solution to which adensity gradient reagent has been added;

(5-2) adding the recovered pancreatic tissue to the purificationsolution in which a density gradient has been set up; and

(5-3) purifying the islets by isolating the islets from the pancreatictissue by centrifugally separating the purification solution to whichthe pancreatic tissue has been added.

Item 17: An isolation method according to Item 16, wherein step (5-1) isa step (5-1′) comprising:

(5-1′) setting up a density gradient in a purification solutioncomprising a tissue protection or preservation solution to which atrypsin inhibitor and a density gradient reagent have been added.

Item 18: An isolation method according to Item 16 or 17, wherein thedensity gradient reagent is iodixanol.

Item 19: An isolation method according to any of items 15 to 18 thatfurther contains the following step comprising:

(1-2) preserving the pancreas into which the protection or preservationsolution has been injected, in a container in which there are formed twolayers: a layer comprising perfluorocarbon and a layer comprising atissue protection or preservation solution that contains a trypsininhibitor.

Item 20: An isolation method according to any of Items 15 to 19, whereinthe trypsin inhibitor is ulinastatin.

Item 21: An isolation method according to any of Items 15 to 20, whereinthe potassium concentration of the tissue protection or preservationsolution is 4 to 50 mmol/L.

Item 22: A pancreatic islet purification solution comprising a tissueprotection or preservation solution that has a potassium concentrationof 4 to 50 mmol/L and to which iodixanol has been added.

Item 23: A purification solution according to item 22, to which atrypsin inhibitor has further been added.

Item 24: A purification solution according to item 23, wherein thetrypsin inhibitor is ulinastatin.

The present invention is described in more detail in the following.

I. Pancreatic Islet Isolation Method

The pancreatic islet isolation method of the present invention comprisesthe steps of:

(1) injecting a protection solution containing a protease inhibitor intothe pancreatic duct of an procured pancreas;

(3) injecting an enzyme solution into the protectionsolution-injected-pancreas and digesting the pancreas; and

(4) purifying the digested pancreatic tissue using a purificationsolution containing a density gradient reagent.

The present invention also encompasses the method that further containsthe step of:

(2) preserving the protection-solution-injected-pancreas using thetwo-layer method.

I(1). Step of Injecting a Protection Solution into the Pancreatic Duct

The isolation method of the present invention includes a step ofinjecting a protease-inhibitor-containing protection solution into thepancreatic duct of a pancreas that has been procured from the donor. Byinjecting the protease-inhibitor-containing preservation solution intothe pancreatic duct, the pancreatic tissue is appropriately protectedand the yield of transplantable islets is increased

The volume of protection solution injected into the pancreatic duct canbe established as appropriate according to the state of the organ or thelike and is approximately 0.1 to 10 ml, preferably approximately 0.1 to2 ml, and more preferably approximately 1 to 1.5 ml, in each case per 1gram organ weight.

The injection of such amounts is preferred from the standpoint ofenabling an appropriate perfusion of the protection solution into thepancreatic duct of the entire pancreas and thereby raising the yield ofgood quality islets.

The protease inhibitor can be selected as appropriate, but at least oneselected from the group consisting of ulinastatin, gabexate mesilate,and nafamostat mesilate is particularly preferred from the standpoint ofobtaining an even better islet yield.

The protection solution preferably has a low potassium concentrationfrom the standpoint of improving the yield of transplantable islets. Inspecific terms, a potassium concentration of 4 to 50 mM, particularly 10to 50 mM, per 1000 mL, protection solution is preferred.

The protection solution preferably further contains trehalose from thestandpoint of increasing the yield of transplantable islets.

The timing of protection solution injection can be established asappropriate, but is preferably as soon as possible and is preferablyimmediately after procurement of the pancreas.

The method of injecting the protection solution can be established asappropriate; for example, a catheter can be inserted into the procuredpancreas and injection can be carried out through this catheter using apump while regulating the injection pressure.

The number of inserted catheters can be selected as appropriate, butpreferably a single catheter is employed. The use of a single catheterminimizes leakage of the solution injected into the organ and enables amore precise injection of the solution and can also reduce damage to theorgan.

I(2). Step of Preservation Using the Two-Layer Method

After the protection solution has been injected into the pancreaticduct, the pancreas is preferably preserved using the two-layer method.

This two-layer method can be implemented by introducing a liquidperfluorocarbon and a preservation solution into a container so as toform two layers; then feeding oxygen into the container; and preservingthe pancreas in an immersed state in the container. The proportionbetween the liquid perfluorocarbon and preservation solution is about1:1 as a volume ratio. The oxygen feed is preferably carried out for atleast 30 minutes.

Preservation of the pancreas by the two-layer method enables a highlevel of tissue viability to be maintained.

The preservation solution used in the two-layer method preferablycontains a protease inhibitor. The islet yield can be increased throughthis use of a protease-inhibitor-containing preservation solution.

The protease inhibitor can be selected as appropriate, but at least oneselected from the group consisting of ulinastatin, gabexate mesilate,and nafamostat mesilate is particularly apt from the standpoint ofobtaining an even better islet yield.

The preservation solution preferably has a low potassium concentration.In specific terms, the potassium concentration is preferably about 4 to50 mmol/L and particularly preferably about 10 to 50 mmol/L.

The use of a preservation solution with a low potassium concentrationenables a further increase in the islet yield to be obtained.

Additionally, the preservation solution preferably further containstrehalose. The use of a preservation solution that contains trehaloseenables a further increase in the islet yield to be obtained.

I(3). Step of Digesting the Pancreas

Digestion of pancreatic tissue is then carried out on the pancreas intowhich protection solution has been injected or on the pancreas that hasbeen preserved by the two-layer method.

Digestion, i.e. degradation, can be carried out, for example, bydistending the pancreas by injecting an enzyme solution into the duct ofthe pancreas and then raising the temperature of the enzyme solution inorder to effect enzyme activation.

Enzyme solution injection can be carried out, for example, by injectingthe enzyme solution into the main pancreatic duct using a pump whilecontrolling the injection pressure. The enzyme solution can be injectedthrough the same catheter as used for injection of the protectionsolution.

As the enzyme solution, a collagenase solution can be used, for example.

After the enzyme solution has been injected into the pancreas, digestioncan be started by raising the temperature of the solution using asuitable device.

For example, when a collagenase solution is employed, the distendedpancreas is placed in a Ricordi chamber; the digestion circuit is filledwith the solution; and the system is closed. The solution is circulatedby a pump and the temperature of the solution is raised to around bodytemperature at about 37° C. The collagenase injected into the pancreatictissue is activated when the temperature is raised, resulting indigestion of the pancreatic tissue through the dissolution of collagen,which forms a tissue that binds the cells to each other.

Digestion is halted at the point at which just the pancreatic exocrinetissue has been dissociated from around the islets while the cellsmaking up the islets remain intact in their aggregated state.

Digestion can be halted by lowering the temperature of the solution.Digestion can also be halted by deactivating the enzyme by adding serumprotein.

For example, digestion can be halted by converting the circulation pathto an open system and passing a room-temperature solution containinghuman albumin through the circulation path. The passage of aroom-temperature solution can lower the solution temperature and canalso dilute the enzyme. The activity of the enzyme can also be reducedby the addition of serum protein.

The pancreatic tissue is recovered after digestion has been stopped. Therecovered pancreatic tissue is preferably centrifugally washed andconcentrated with a centrifugal separator prior to purification.

I(4). Purification Step

Purification can be carried out utilizing the fact that the islets havea lighter specific gravity than pancreatic exocrine tissue. For example,the digested pancreatic tissue can be added to a solution in which adensity gradient, i.e., a specific gravity concentration gradient, hasbeen formed and the islets can be purified by separation of the isletsfrom the pancreatic exocrine tissue by density gradient andcentrifugation.

The following procedure, for example, can be used.

First, a density gradient is formed in a purification solution thatcontains a density gradient reagent.

Then, the digested pancreatic tissue is added to the purificationsolution in which the density gradient is formed.

The purification solution loaded with the digested pancreatic tissue isthen subjected to centrifugal separation by density gradient andcentrifugation. Separating the islets from the exocrine tissue by thiscentrifugal separation, and the islets are isolated from the recoveredpancreatic tissue.

A density gradient reagent that can form a low viscosity solution ispreferred. Suitable density gradient reagents include Iodixanol(Optiprep™) andN,N′-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)acetoamido]-2,4,6-triiodo-isophthalamide)(Nycodenz®).

The purification solution preferably additionally contains a proteaseinhibitor. A purification solution containing a protease inhibitor canfurther improve the islet yield.

The density gradient may be either a continuous density gradient or adiscontinuous density gradient; however, a continuous density gradientis preferred because it enables the recovery of more islets.

The density gradient can be formed as appropriate by known methods.Appropriate apparatus can be used, for example, an instrument that formsa continuous density gradient.

A cell processor, such as a COBE 2991, can also be utilized forpurification.

For example, a density gradient can be formed in the COBE 2991 using adensity gradient reagent; the digested pancreatic tissue, after washingand concentration, can be added thereto; and, after the islets have beenseparated from the exocrine tissue by continuous density gradient andcentrifugation, the solution in the COBE 2991 can be recovered inindividual fractions. After separation, the microscopic inspection ofthe solution is carried out to determine which fractions contain theislets and recover the islets.

This recovery of the purified islets completes the series of isolationsteps.

II. Protection Solution for Injection into Pancreatic Duct

The protection solution of the present invention can be used forinjection into the pancreatic duct during the isolation of islets forislet transplantation. In addition, the protection solution of thepresent invention can be very suitably used as theprotease-inhibitor-containing protection solution in the isolationmethod of the present invention described above.

The protection solution of the present invention can be obtained byadding a protease inhibitor to a tissue protection or preservationsolution.

II-1. Protease Inhibitor

The protease inhibitor used in the present invention is not particularlylimited by its origin and type, as long as it has protease-inhibitingactivity. Known protease inhibitors can be suitably used, but a trypsininhibitor having a trypsin-inhibiting activity is particularlypreferred.

Examples of protease inhibitors include at least one selected from thegroup consisting of ulinastatin (Miraclid™),4-[2-aminoethylbenzenesulfonyl]fluoride (AEBSF, Pefabloc™), gabexatemesilate (FOY™), and nafamostat mesilate (Fusan™). Among these, at leastone selected from the group consisting of ulinastatin, gabexatemesilate, and nafamostat mesilate is preferred for use from thestandpoint of further improving the islet yield. Ulinastatin andgabexate mesilate are also preferred for their anti-inflammatoryactivity.

The rate of addition of the protease inhibitor to the protectionsolution can be established as appropriate in accordance with the typeof inhibitor and within a range in which the effects of the presentinvention are achieved. When the protease inhibitor is ulinastatin, therate of addition is about 10,000 to 100,000 U per liter and preferablyabout 50,000 to 100,000 U per liter. When the protease inhibitor isgabexate mesilate, the rate of addition is about 100 to 10,000 mg perliter and preferably about 500 to 2,000 mg per liter.

II-2. Tissue Protection or Preservation Solution

The tissue protection or preservation solution can be selected suitablyfrom known solutions used for the protection or preservation of tissue(including organs and cells).

For example, Euro-Collins solution, University of Wisconsin solution (UWsolution), ET-Kyoto solution, Low-Potassium Dextran Glucose solution,HTK solution (Custodiol™), and so forth can be used.

Solutions with the compositions given below can also be provided asexamples.

Tissue Protection or Preservation Solution Example 1

Sodium (Na⁺) 10 to 140 mmol/L Potassium (K⁺) 4 to 140 mmol/L Magnesium(Mg²⁺) 0 to 4 mmol/L Calcium (Ca²⁺) 0 to 2 mmol/L Phosphate (H₂PO₄ ⁻ orHPO₄ ²⁻) 12 to 65 mmol/L Cl⁻, HCO₃ ⁻, CO₃ ²⁻, organic acid, 15 to 150mmol/L or organic acid anion Hydroxyl ethyl starch 0 to 80 g/L Trehalose0 to 240 mmol/L

Tissue Protection or Preservation Solution Example 2

Sodium (Na⁺) 10 to 140 mmol/L Potassium (K⁺) 4 to 140 mmol/LHydroxyethyl starch 0 to 80 g/L Glutathione 0 to 10 mmol/L Adenosine 0to 10 mmol/L Lactobionate 0 to 140 mmol/L Raffinose 0 to 50 g/L

Tissue Protection or Preservation Solution Example 3

Sodium (Na⁺) 80 to 120 mmol/L Potassium (K⁺) 4 to 50 mmol/L Gluconate 15to 150 mmol Phosphate 20 to 40 mmol/L Trehalose 80 to 160 mmol/L (27 to55 g/L) Hydroxyl ethyl starch (HES) 20 to 60 g/L Dibutyryl cAMP 0 to 10mmol/L Nitroglycerin 0 to 1 g/L

Among the tissue protection or preservation solutions provided asexamples above, the ET-Kyoto solution, the solution described in example1, and the solution described in example 3 are particularly preferred.

II-3. Embodiments of the Protection Solution for Injection intoPancreatic Duct

The protection solution for injection into the pancreatic ductpreferably has a low potassium concentration. In specific terms, apotassium concentration of 4 to 50 mM, particularly 10 to 50 mM per 1000mL protection solution is preferred.

The use of a low potassium concentration avoids the induction ofvasospasm and can thereby prevent insulin release from the islets andenable the solution to quickly spread throughout the tissue. Thisresults in an even higher pancreatic duct protection action and improvesthe yield of transplantable islets.

The osmolarity of the protection solution for injection into thepancreatic duct is preferably 270 to 450 mOsm/L and particularlypreferably is 300 to 400 mOsm/L. Swelling or shrinkage of the tissueduring protection or preservation can be prevented within this range.

The pH of the protection solution for injection into the pancreatic ductis preferably about 7 to 8 in order to stop acidic degradation of thecells and tissue.

The protection solution for injection into the pancreatic ductpreferably contains trehalose. The presence of trehalose provides anadditional increase in protective action on the pancreatic duct and canthereby improve the islet yield. Trehalose exists in three forms, i.e.α,α-trehalose, α,β-trehalose, and β,β-trehalose; any of these may beused and their mixtures may be used. α,α-Trehalose, present in nature,is preferably used.

Trehalose concentration is about 0 to 400 mmol, particularly about 50 to240 mmol, and even more particularly about 80 to 160 mmol, in each caseper 1000 mL of the protection solution.

The protection solution for injection into the pancreatic duct may alsocontain other components insofar as the effects of the present inventionare not impaired. These other components can be exemplified by variouselectrolytes, sugars, amino acids, drugs, vitamins, and so forth.

The protection solution for injection into the pancreatic duct may alsocontain a cell activator such as AMP (e.g., dibutyryl cAMP) or ATP, avasodilator such as prostaglandin or nitroglycerin, antibiotics,adenosine, N-acetyl-L-cysteine, glycine, ascorbic acid, glutamine,nicotinamide, glutathione, raffinose, and so forth.

A solution containing at least the following components in the followingproportions is an example of a preferred embodiment of the protectionsolution according to the present invention for injection into thepancreatic duct.

Sodium 80 to 120 mmol/L Potassium 4 to 50 mmol/L Gluconate 15 to 150mmol/L Phosphate 20 to 40 mmol/L Trehalose 80 to 160 mmol/L Hydroxylethyl starch (HES) 20 to 60 g/L Ulinastatin 10,000 to 100,000 U/L

The protection solution according to the present invention for injectioninto the pancreatic duct has a high protective action on the pancreaticduct and can keep the tissue viablity and can stabilize andsatisfactorily protect the tissue and as a result can raise the yield oftransplantable islets.

III. Pancreas Preservation Solution for the Two-Layer Method

The preservation solution according to the present invention can be usedfor pancreas preservation by the two-layer method during the isolationof islets for islet transplantation. In addition, the pancreaspreservation solution according to the present invention for thetwo-layer method can be very suitably used as the preservation solutionused in the two-layer method in the islet isolation method of thepresent invention described above.

The preservation solution according to the present invention can beobtained by the addition of a protease inhibitor to a tissue protectionor preservation solution that has a low potassium concentration.

The inhibitors described in II-1 above can be used as the instantprotease inhibitor.

The low-potassium versions of the solutions described in II-2 above canbe used as the tissue protection or preservation solution. ET-Kyotosolution is particularly preferred.

III-2. Embodiments of Pancreas Preservation Solution for the Two-LayerMethod

The amount of protease inhibitor to be added to the preservationsolution can be established as appropriate in accordance with the typeof inhibitor and within a range in which the effects of the presentinvention are achieved.

When the protease inhibitor is ulinastatin, the amount to be added isabout 10,000 to 100,000 U per liter and preferably about 50,000 to100,000 U per liter. When the protease inhibitor is gabexate mesilate,the amount to be added is about 100 to 10,000 mg per liter andpreferably about 500 to 2000 mg per liter.

The potassium concentration in the pancreas preservation solution forthe two-layer method is about 4 to 50 mM and particularly 10 to 50 mM.

The use of a low potassium concentration provides an even moresatisfactory pancreas preservation activity and can thereby improve theyield of transplantable islets.

The osmolarity of the preservation solution is preferably 270 to 450mOsm/L and is particularly preferably 300 to 400 mOsm/L. Swelling orshrinkage of the tissue during preservation can be prevented within thisrange.

The pH of the preservation solution is preferably about 7 to 8 in orderto prevent acidic degradation of the cells and tissue.

The preservation solution preferably contains trehalose.

Trehalose concentration is about 0 to 400 mmol, particularly about 50 to240 mmol, and even more particularly about 80 to 160 mmol, in each caseper 1000 mL the preservation solution. The presence of trehaloseprovides an additional increase in pancreas protection activity and canthereby increase the islet yield.

The preservation solution may also contain other components insofar asthe effects of the present invention are not impaired. These othercomponents can be exemplified by various electrolytes, sugars, aminoacids, drugs, vitamins, and so forth.

The preservation solution may also contain a cell activator such as AMP(e.g., dibutyryl cAMP) or ATP, a vasodilator such as prostaglandin ornitroglycerin, antibiotics, adenosine, N-acetyl-L-cysteine, glycine,ascorbic acid, glutamine, nicotinamide, glutathione, raffinose, and soforth.

A solution containing at least the following components in the followingproportions is an example of a preferred embodiment of the preservationsolution according to the present invention for use in the two-layermethod.

Sodium 80 to 120 mmol/L Potassium 4 to 50 mmol/L Gluconate 15 to 150mmol/L Phosphate 20 to 40 mmol/L Trehalose 80 to 160 mmol/L Hydroxylethyl starch (HES) 20 to 60 g/L Ulinastatin 10,000 to 100,000 U/L

Use of the preservation solution according to the present inventionprovides a highly protective action on the pancreas and can keep thetissue viability intact and can satisfactorily preserve the tissuesthereof, and as a result can substantially improve the yield oftransplantable islets.

IV. Islet Purification Solution

The purification solution of the present invention is well-adapted forislet purification in islet isolation during islet transplantation. Inaddition, the purification solution according to the present inventionis well-adapted for use as the purification solution in the isletisolation method according to the present invention described above.

The purification solution according to the present invention can beobtained by adding a density gradient reagent to a tissue protection orpreservation solution that contains trehalose. The trehalose-containingversions of the solutions described in II-2 above can be used as thistrehalose-containing tissue protection or preservation solution. The useof ET-Kyoto solution is particularly preferred.

IV-1. Density Gradient Reagent

The density gradient reagent can be selected as appropriate from knowndensity gradient reagents used to prepare a density gradient in asolution. A density gradient reagent that can form a low-viscositysolution is particularly preferred. It is also preferred that thedensity gradient reagent have a low endotoxin level.

Suitable examples of the density gradient reagent include iodixanol(Optiprep™) andN,N′-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)acetoamido]-2,4,6-triiodo-isophthalamide(Nycodenz®). Iodixanol is particularly suitable.

Using these as the density gradient reagent makes it possible to obtaina low-viscosity purification solution and can expedite the purificationspeed. In addition, they can provide solutions with a low endotoxinlevel.

The mixing proportion of the density gradient reagent with respect tothe tissue protection or preservation solution can be established asappropriate by measuring the density of the pancreas tissue prior topurification and considering the specific gravity of the densitygradient reagent and the tissue protection or preservation solution.

IV-2. Trehalose

Trehalose exists in three forms, i.e. α,α-trehalose, α,β-trehalose, andβ,β-trehalose; any of these may be used and their mixtures may be used.α,α-Trehalose, present in nature, is preferably used.

The trehalose concentration in the purification solution is about 0 to400 mmol/L, particularly 50 to 240 mmol/L, and even more particularlyabout 80 to 160 mmol/L.

IV-3. Protease Inhibitor

The purification solution preferably also contains a protease inhibitor.The protease inhibitors described in II-1 above can be used as thisprotease inhibitor. In particular, at least one selected from the groupconsisting of ulinastatin, gabexate mesilate, and nafamostat mesilate ispreferred from the standpoint of further improving the islet yield.

In those cases where a protease inhibitor is added to the purificationsolution, the rate of addition can be established as appropriate inaccordance with the type of inhibitor and within a range in which theeffects of the present invention are achieved.

When the protease inhibitor is ulinastatin, the amount to be added isabout 10,000 to 100,000 U per liter and preferably about 50,000 to100,000 U per liter. When the protease inhibitor is gabexate mesilate,the amount to be added is about 100 to 10,000 mg per liter andpreferably about 500 to 2,000 mg per liter.

IV-4. Embodiments of the Purification Solution

The purification solution preferably has a low potassium concentration,wherein about 4 to 50 mmol/L is preferred and about 10 to 50 mmol/L isparticularly preferred.

The purification solution preferably has a low viscosity. In particular,the viscosity measured by the Brookfield method at a measurementtemperature of 22° C. should be no greater than 5 centipoise (cP),preferably no greater than 3 cP, and more preferably no greater than 2cP.

Other components may be added as appropriate to the purificationsolution insofar as the effects of the present invention are notimpaired. These other components encompass, for example, adenosine,dextran, heparin, and so forth.

An example of a preferred embodiment of the purification solution is asolution containing at least the following components in the followingproportions:

Sodium 80 to 120 mmol/L Potassium 4 to 50 mmol/L Gluconate 15 to 150mmol/L Phosphate 20 to 40 mmol/L Trehalose 80 to 160 mmol/L Hydroxylethyl starch (HES) 20 to 60 g/L Iodixanol 100 to 500 mL/L

An example of a preferred embodiment of theprotease-inhibitor-containing purification solution is a solutioncontaining at least the following components in the followingproportions:

Sodium 80 to 120 mmol/L Potassium 4 to 50 mmol/L Gluconate 15 to 150mmol/L Phosphate 20 to 40 mmol/L Trehalose 80 to 160 mmol/L Hydroxyethylstarch (HES) 20 to 60 g/L Iodixanol 100 to 500 mL/L Ulinastatin 10,000to 100,000 U/L

In islet purification for transplantation, it is crucial that isletpurification be carried out in such a manner that the three-dimensionalstructure of the islets is preserved. The use of the purificationsolution according to the present invention makes it possible to raisethe yield of transplantable islets residing in a state in which thethree-dimensional structure of the islets has been well maintained.

V. Islet Transplantation

Islet transplantation can be carried out by infusing the islets obtainedby the series of isolation steps described above into the portal vein ofa patient.

When the isolated islets satisfy established criteria, they are judgedas suitable for transplantation and are then used for transplantation.

Viewed from the perspective of efficacy, determination as to whether theislets to be used for transplantation will function as isletspost-transplantation is required. In addition, the risk of introducingpathogens, toxic substance, and so forth into the recipient must beexcluded to the maximum extent possible.

In specific terms, the following criteria are used fortransplantation-qualified isolated islets.

Islet Yield≧4,000 IE/kg (patient body weight)

Purity≧30%

Tissue volume≦10 ml

Viability≧70%

Endotoxin≦5 IE/kg (patient body weight)

Negative Gram stain

An islet yield≧5,000 IE/kg (Patient body weight) is more appropriate forcarrying out islet transplantation more efficiently.

Islets that have been evaluated as qualified for transplantation arepreserved until the patient (recipient) can be prepared. Once thepatient has been prepared, administration is carried out into the portalvein by infusion.

As necessary, various known techniques for islet transplantation andislet isolation can be added to the technology according to the presentinvention for islet isolation.

The present invention increases the yield of transplantable islets. Thepresent invention can also raise the efficiency of purification and canimprove the speed of purification. This in turn makes it possible totransplant large numbers of good-quality islets and can bring about amore effective post-transplant islet functionality.

The use of the isolation method and solutions according to the presentinvention can improve clinical outcome resulting from islettransplantation.

Effect of the Invention

The islet isolation method of the present invention increases the yieldof transplantable islets. It also enables islet isolation to be carriedout at efficiently in a shorter period of time.

The islet isolation method of the present invention is characterized bythe injection of a protease-inhibitor-containing protection solutioninto the pancreatic duct.

This injection of a protease-inhibitor-containing protection solutioninto the pancreatic duct provides good protection for the pancreatictissue in the pancreatic duct and thereby improves the yield oftransplantable islets.

The isolation method of the present invention may also incorporate astep of preserving the pancreas using the two-layer method. Preservationusing the two-layer method is preferably carried out using (i) liquidperfluorocarbon and (ii) a preservation solution that contains aprotease inhibitor and that has a potassium concentration of 4 to 50mmol/L. This provides a remarkable improvement in the yield oftransplantable islets.

The isolation method of the present invention further comprises a stepof purification using a purification solution that contains a densitygradient reagent. Purification is preferably carried out using apurification solution that contains a density gradient reagent andtrehalose. Purification is more preferably carried out using apurification solution that further contains a protease inhibitor. Theuse of these purification solutions raises the efficiency ofpurification and also improves the cells of the islets fit fortransplantation.

The isolation method of the present invention, because it has thecharacteristic features cited above, is also characterized by itsability to bring about a substantial increase in the yield oftransplantable islets.

The present invention further provides a protease-inhibitor-containingprotection solution for injection into the pancreatic duct. Thisprotection solution has a highly protective effect upon the pancreaticduct and thereby brings about an increase in the islet yield.

The present invention further provides a pancreas preservation solutionfor the two-layer method, said solution containing protease inhibitorand having a potassium concentration of 4 to 50 mmol/L. Thispreservation solution is highly protective of the pancreas and maintainstissue viability and provides thorough tissue protection, therebyincreasing the yield of transplantable islets.

The present invention further provides an islet purification solutionthat contains a density gradient reagent and trehalose. The presentinvention further provides a purification solution that contains adensity gradient reagent, trehalose, and a protease inhibitor. Thesepurification solutions can raise the efficiency of purification and canalso raise the yield of transplantable islets. The lowered viscosity ofthese purification solutions can boost the speed of purification.Moreover, they have low endotoxin levels and can also lower the risk tothe patient.

Thus, the present invention provides an islet isolation method, aprotection solution for injection into the pancreatic duct, a pancreaspreservation solution for the two-layer method, and an isletpurification solution, each of which can raise the yield oftransplantable islets.

The present invention raises the yield of islets that satisfy thequalifying criteria for transplantation and as a consequence canincrease the success rate of islet transplantation. Moreover, thepresent invention makes it possible to realize islet transplantationfrom non-heart-beating donors, which in fact has not been possible withprior-art methods.

As a consequence of the preceding, the present invention can provide amore reliable and more effective treatment for insulin-dependentdiabetics and particularly type-I diabetic patients and substantiallycontributes to the clinical implementation of islet transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an experiment that compared islet yield withprotection of the pancreatic duct against the islet yield withoutprotection of the pancreatic duct. The islet yield before purificationis shown in FIG. 1A. The islet yield after purification is shown in FIG.1B. IE/g indicates the mean number of islets yielded per 1 grampancreas. Ductal injection (−) shows the case in which protectionsolution was not injected into the pancreatic duct, while ductalinjection (+) shows the case in which protection solution was injectedinto the pancreatic duct.

FIG. 2 shows the results of an experiment that compared the islet yieldafter purification for the use of different preservation solutions inthe two-layer method. IE/pancreas shows the total yield of islets takenfrom a single pancreas. PFC refers to liquid perfluorocarbon (PFC); UWrefers to UW solution; M-UW refers to UW+ulinastatin solution; ET-Kyotorefers to ET-Kyoto solution; and M-Kyoto refers to ET-Kyoto+ulinastatinsolution.

FIG. 3 shows the results for islet yield in porcine islet isolation witha comparison of individual protocols. The pre-purification results areshown in FIG. 3A. The post-purification results are shown in FIG. 3B.IE/g indicates the mean number of islets yielded per 1 gram pancreas.

FIG. 4A shows the change in the HbA1c value for 6 patients pre- andpost-transplantation from non-heart-beating donors. A different symbolis used to show the data for each patient. The y-axis in FIG. 4A showsthe percentage HbA1c with reference to the total hemoglobin. The dashedline shows the upper limit for normal values.

FIG. 4B shows the C-peptide value (mean value) in the glucagonstimulation test. The filled square (▪) shows the basal value and theopen square (□) shows the value after stimulation.

FIG. 5A shows the results of measurement before and after islettransplantation of blood glucose value before breakfast and beforedinner. The two dashed lines in FIG. 5A indicate the normal range. Thefilled diamonds (♦) show the values before breakfast, while the filledsquares (▪) show the values before dinner.

FIG. 5B shows the daily amount of insulin for a patient before and afterislet transplantation.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in the following, using examples andexperimental examples in order to further elucidate the presentinvention; however, the present invention is not limited to theseexamples.

Materials and Methods 1. Materials 1(1): Protection Solutions andPreservation Solutions

UW solution (ViaSpan™, made by DuPont) and ET-Kyoto solution (KyotoSolution, Kyoto Biomedical Science Co., Ltd.) were used.

ET-Kyoto+ulinastatin solution (hereinafter also referred to M-Kyotosolution) was also prepared by adding 100,000 units (U) ulinastatin(Miraclid™, Mochida Pharmaceutical Co., Ltd.) to 1 L ET-Kyoto solution.

The composition of the main components present in UW solution, ET-Kyotosolution, and M-Kyoto solution and the osmolarity are given in Table 1.

TABLE 1 ET-Kyoto M-Kyoto UW Solution Solution Solution Sodium (Na) 29100 100 (mmol/L) Potassium (K) 125 43.5 43.5 (mmmol/L) Magnesium (Mg) 5— — (mmol/L) Gluconate — 100 100 (mmol/L) Phosphate 25 25 25 (mmol/L)Sulfate 5 — — (mmol/L) Lactobionate 100 — — (mmol/L) Raffinose 30 — —(mmol/L) Trehalose — 120 120 (mmol/L) Adenosine 5 — — (mmol/L)Alloprinol 1 — — (mmol/L) Glutathione 3 — — (mmol/L) Hydroxy Ethyl 50 3030 Starch (HES) (g/L) Ulinastatin (×10³ U/L) — 100 Osmorality 320 366366 (mOsm)

ET-Kyoto+Pefabloc solution was prepared by adding 0.4 to 8 mM4-[2-aminoethylbenzenesulfonyl]fluoride.HCl (AEBSF, Pefabloc™, made byRoche Diagnostics) to 1 L ET-Kyoto solution.

ET-Kyoto+FOY solution was prepared by adding 1000 mg gabexate mesilate(FOY™, made by Ono Pharmaceutical Co., Ltd.) to 1 L ET-Kyoto solution.

1(2). Purification Solutions

Ficoll solution was prepared by mixing two Ficoll solutions (Ficoll™,Pharmacia Corporation) having different concentrations (dilution withHanks' solution) so as to provide a light specific gravity of about 1.07to 1.08 and a heavy specific gravity of 1.09 to 1.12.

ET-Kyoto+iodixanol solution (hereinafter also referred to as I-Kyotosolution) was prepared by mixing a 60 weight % aqueous iodixanolsolution (OptiPrep™, made by AXIS-SHIELD) with ET-Kyoto solution.

ET-Kyoto+ulinastatin+iodixanol solution (hereinafter also referred to asMI-Kyoto solution) was prepared by mixing 60 weight % aqueous iodixanolsolution (OptiPrep™, made by AXIS-SHIELD plc) with M-Kyoto solution.ET-Kyoto+Pefabloc+iodixanol solution (hereinafter also referred to asKyoto+AEBSF+Idx solution) was prepared by mixing4-[2-aminoethylbenzenesulfonyl]fluoride.HCl (Pefabloc™, made by RocheDiagnostics) and 60 weight % aqueous iodixanol solution (OptiPrep™, madeby AXIS-SHIELD plc) with ET-Kyoto solution.

The ET-Kyoto solution had a specific gravity of about 1.04 and theiodixanol solution had a specific gravity of approximately 1.32. Whenthese were mixed, a light specific gravity of about 1.07 to 1.08 and aheavy specific gravity of 1.09 to 1.12 were prepared by changing theratio between the two solutions.

2. Procedure for Porcine Islets Isolation

Porcine islets were isolated using the following procedure.

Porcine pancreatic tissues were obtained at a slaughterhouse in Kyoto.After pancreas procurement, (1) the pancreases were immediatelypreserved in a container in which two layers had been formed by thetwo-layer method or (2) a catheter was inserted into each pancreasimmediately after procurement and pancreatic duct protection solutionwas injected through the catheter into the main pancreatic duct of eachpancreas and the pancreases were thereafter quickly preserved in acontainer in which two layers had been formed by the two-layer method,the two layers being PFC and preservation solution. The volume ofpancreatic duct protection solution injected in (2) was 1 mL per 1 grampancreas weight.

Here, the time from heart beat cessation to immersion of the pancreas inthe preservation solution is defined as the warm ischemic time. The timefrom immersion of the pancreas in the preservation solution to the startof islet separation is defined as the cold ischemic time.

The pancreas preserved by the two-layer method was transported to theislet isolation facility at the University of Kyoto and was thendecontaminated.

A cold collagenase solution (Liberase HI™, made by Roche MolecularBiochemicals) was then injected into the main duct of the pancreas andthe pancreas was distended while the solution was circulated.

The distended pancreas was cut into nine pieces and placed in a Ricordichamber. The circulation path in this chamber was filled with solutionand the system was closed. Then, while circulating the solution with apump, the temperature of the solution was raised to about 37° C. toactivate the collagenase and the pancreatic tissue was digested byrepeatedly circulating the collagenase solution through the Ricordichamber. The digestion process was stopped at the point at which justthe pancreatic exocrine tissue had been dissociated from around theislets while the cells making up the islets remained intact in theiraggregated state.

To stop digestion, the collagenase was deactivated by lowering thetemperature of the solution by opening the circulation path and passinga human-albumin-containing solution at room temperature through thecirculation path.

The pancreatic tissue was recovered after digestion had been halted, bypassage of the room temperature solution through the circulation path.

The time from placement of the pancreas in the Ricordi chamber to thestart of recovery of the digested pancreatic tissue was designated asphase 1.

The time from the start of recovery until recovery was complete wasdesignated as phase 2.

The recovered pancreatic tissue was centrifugally washed with acentrifugal separator and was aggregated and concentrated.

A continuous density gradient was formed in a blood cell washinginstrument (COBE 2991 cell processor, Gambro BCT) using a purificationsolution that contained a density gradient reagent.

The washed and concentrated pancreatic tissue was then added to thesolution in which the density gradient had been formed.

Purification was carried out by isolating the islets from the pancreatictissue by carrying out centrifugal separation using the continuousdensity gradient and centrifugation technique to separate the isletsfrom the pancreatic exocrine tissue. After this, the solution in theblood cell washing instrument was collected into individual fractionsand a microscopic examination administered to determine which fractionsthe islets were present in, and the islets were then recovered.

3. Evaluation Methods

The islet yield and purity were evaluated by dithizone staining. Afterdithizone staining a 100 μL sample was taken from the solution suspendedin 200 mL and the islet yield was evaluated under a microscope byindividual sizes. The purity was given by the proportion of the isletsin the total mass inclusive of all elements, e.g., islets, exocrinetissue in addition to the islets, pancreatic duct, and so forth.

The islet size was obtained as an average of the islets enumerated byindividual sizes.

The efficiency of purification was obtained by dividing the number ofislets after purification by the number of islets before purification.

The morphological score (gross morphological evaluation) wasqualitatively assessed by having two investigators score the following:shape (flat versus spherical), border (irregular versus well-rounded),integrity (fragmented versus solid/compact), staining quality (uniformversus non-uniform), and diameter (all<100 μL versus>10%>200 μL). Eachparameter was scored from 0 to 2 with 0 being the lowest score and 2being the highest score. The lowest total score for islet isolation wastherefore 0 and the highest was 10. Spherical, well-rounded, solid orcompact, uniformly stained, and large diameter islets were characterizedand scored as preferred islets.

Islet viability was assessed by the simultaneous visualization of liveand dead cells using acridine orange (10 μmol/L) and propidium iodide(15 μmol/L) (AO/PI). 50 islets were investigated and the viability ineach one was visually determined and the mean thereof was calculated.

The stimulation index was calculated from the ratio between insulinsecretion at a high glucose concentration to that at a low glucoseconcentration.

Islet function was assessed according to the method of Shapiro andColleagues by monitoring the insulin secretion response of the purifiedislets to glucose challenge. In brief, 100 Islet Equivalents (IE) wereincubated on CMRL solution at 37° C. in a 5% CO₂ atmosphere and thenincubated for 2 hours at 37° C. in a 5% CO₂ atmosphere on RPMI1640solution (GIBCO BRL) containing 2.8 mM ether or 20 mM ether and 20 mMglucose; the supernatant was recovered and the insulin value wasmeasured with an ELISA kit (Morinaga Biochemical Industry Co., Ltd.)using an antigen-antibody reaction.

The values obtained in the examples and experimental examples werereported using a mean and standard deviation (mean±SE). Comparisons weremade among the three groups using the mean of ANOVA and Fisher's PLSDpost-hoc test. Values with a P value less than 0.05 were taken assignificant.

Example 1 Investigation of Pancreatic Duct Protection

Comparative experiments were carried out using the procedure describedabove in order to carry out a comparative investigation of thepresence/absence of pancreatic duct protection. The procedures wereidentical, except that after pancreas procurement, (1) preservation bythe two-layer method was immediately carried out (no pancreatic ductprotection) or (2) protection solution was injected into the main ductof the pancreas immediately after procurement followed by preservationby the two-layer method (implementation of pancreatic duct protection).

In these experiments, M-Kyoto solution was used as the pancreatic ductprotection solution. M-Kyoto solution and liquid PFC were used in thetwo-layer method. Ficoll solution was used as the purification solution.

The islet yields obtained in these experiments before and afterpurification are shown in FIG. 1 (1A: Before purification, 1B: Afterpurification) and the following table. IE/g denotes the mean number ofislets per 1 gram pancreas.

TABLE 2 Islet Yield (IE/g) Before Purification After Purification NoPancreatic Duct 6,889 ± 749 3,662 ± 320  Protection (−) Implementationof Pancreatic 10,626 ± 2153 4,807 ± 1227 Duct Protection (+)

As shown in Table 2, both before purification and after purification, itwas demonstrated that the islet yield with pancreatic duct protection(implementation of pancreatic duct protection) was significantly higher,than in the absence of pancreatic duct protection.

The other characteristics of the islets are shown in Table 3 below.

TABLE 3 Implementation of No Pancreatic Duct Pancreatic Duct Protection(−) Protection (+) Viability 95.3 ± 1.5 96.2 ± 1.2 Morphological score 7.4 ± 0.7  7.8 ± 0.6 Purity (%) 86.3 ± 3.8 91.3 ± 4.3 Stimulation Index 1.7 ± 0.3  2.6 ± 1.4

As shown in Table 3, no significant difference was seen between the twogroups with regard to the other islet characteristics.

Example 2 Comparative Investigations on Preservation by the Two-LayerMethod

In order to perform comparative investigations of the use of differentsolutions in the two-layer method, comparative investigations werecarried out using the same procedure, but using the different solutionsshown in Table 4 for the two-layer method. The M-UW solution wasobtained by adding 100,000 units (U) ulinastatin to 1 L UW solution.

The experiments were carried out using the following procedure.

Islets were isolated from inbred male Lewis rats weighing from 300 to380 grams (Chaeles River Laboratories, Wilmington, Mass.). The ratstudies were approved by the Review Committee of the Kyoto UniversityGraduate School of Medicine. The common bile duct was cannulated using a24-gauge catheter (Baxter, Deerfield, Ill.) and fixed and the end wasthen clamped. The pancreas, spleen, and duodenum were removed en blocand preserved in the particular solution. The pancreas was preserved for6 hours at 4° C. The islets were isolated using the modified method ofSawada (Transplantation. 2003; 75(12): 1965-1969). After preservation,each pancreas was distended using a 2 mg/mL collagenase solution (24 mgServa collagenase, Serva, Heidelberg, Germany) in 12 mL Hanks' BalancedSalt Solution (HBSS). The spleen and duodenum were removed and thepancreas was incubated for 22 minutes at 37° C. in a 50 mL conical tubewithout shaking. The digested pancreas was washed three times with UWsolution by centrifugal separation (150 g, 3 minutes, 8° C.). It wasthen purified with a discontinuous density gradient (1.030, 1.095,1.105, 1.125 g/cm³) formed using a solution of iodixanol (Optiprep™,Nycomed Pharma AS, Oslo, Norway) added to M-Kyoto solution. Theresulting islet yield was assessed by dithizone staining.

The results of the post-purification islet yield are shown in FIG. 2 andTable 4. IE/pancreas denotes the total islet yield taken from eachindividual pancreas.

TABLE 4 Solution Used in the Post-Purification Islet Yield Two-LayerMethod (IE/pancreas) UW/PFC 622 ± 133 M-UW/PFC 730 ± 114 ET-Kyoto/PFC700 ± 116 M-Kyoto/PFC 1207 ± 76 

As shown in Table 4 and FIG. 2, the post-purification islet yield wasfound to be significantly higher with the use of M-Kyoto solution forpreservation in the two-layer method.

Example 3 Investigation of the Isolation Protocol 3-1. Protocol Outline

A comparative investigation was carried out using the following threeisolation protocols in the porcine islet isolation procedure describedabove.

C1 Protocol

Injection of protection solution into the pancreatic duct was notcarried out.

UW solution and liquid perfluorocarbon (PFC) were used in the two-layermethod.

Ficoll solution was used as the purification solution.

C2 Protocol

Injection of protection solution into the pancreatic duct was carriedout and pancreatic duct protection was implemented. M-Kyoto solution wasused as the protection solution.

M-Kyoto solution and liquid perfluorocarbon (PFC) were used in thetwo-layer method.

Ficoll solution was used as the purification solution.

Kyoto Protocol

Injection of protection solution into the pancreatic duct was carriedout and pancreatic duct protection was implemented. M-Kyoto solution wasused for the protection solution.

M-Kyoto solution and liquid perfluorocarbon (PFC) were used in thetwo-layer method.

I-Kyoto solution was used as the purification solution.

The features of each protocol are given in Table 5.

TABLE 5 Pancreatic Duct Solutions Used in the Purification ProtocolProtection Two-Layer Method Solution C1 protocol No injection of UW/PFCFicoll protection solution C2 protocol Injection of M-Kyoto solution/Ficoll M-Kyoto solution PFC Kyoto Injection of M-Kyoto solution/ I-Kyotoprotocol M-Kyoto solution PFC solution

3.2. Comparison of Protocols 3.2(1). Characteristics of the IsolationProcesses

The characteristics of the isolation process are shown in Table 6 foreach protocol.

TABLE 6 C1 Protocol C2 Protocol Kyoto Protocol Pancreas Size (g) 104 ±13  105 ± 8  104 ± 8  Operation Time (min) 9 ± 1 13 ± 1 13 ± 1 WarmIschemic Time (min) 19 ± 1* 24 ± 2 25 ± 1 Cold Ischemic Time (min) 148 ±9** 120 ± 0  120 ± 0  Phase 1 Period (min)  13 ± 1***  8 ± 1  8 ± 1Phase 2 Period (min) 27 ± 1  38 ± 4 34 ± 3 The * in the table indicatesthat the warm ischemic time for the C1 protocol was significantlyshorter than the C2 protocol (P < 0.01) and the Kyoto protocol (P <0.01). The ** in the table indicates that the cold ischemic time for theC1 protocol was significantly longer than for the C2 protocol (P < 0.01)and the Kyoto protocol (P < 0.01). The *** in the table indicates thatphase 1 for the C1 protocol was significantly longer than for the C2protocol (P < 0.02) and the Kyoto protocol (P < 0.01).

There was no substantial difference in pancreas size or operation timeamong the three groups.

The C1 protocol, because it lacked the step of injection into thepancreatic duct, had a significantly shorter warm ischemic time than theother protocols. However, the C1 protocol had a significantly longercold ischemic time than the other protocols because it required catheterinsertion.

Phase 1 was particularly long with the C1 protocol. However, phase 2 wasabout the same among the three groups.

3-2(2). Islet Yields

The islet yields before and after purification in the individualisolation protocols are shown in Table 7 below and in FIGS. 3A and 3B.IE/g denotes the mean number of islets yielded per 1 gram pancreas.

TABLE 7 Islet Yield (IE/g) Before Purification After Purification C1Protocol 4,809 ± 454 IE/g 2,486 ± 394 IE/g C2 Protocol 8,846 ± 904 IE/g3,527 ± 795 IE/g Kyoto Protocol 10,247 ± 637 IE/g  7,253 ± 915 IE/g

As shown in Table 7 and FIG. 3, the values for the islet yield beforepurification were significantly higher in the C2 protocol and the Kyotoprotocol than in the C1 protocol (FIG. 3A).

In addition, the value of the islet yield after purification wassignificantly higher in the Kyoto protocol than in the C1 protocol andC2 protocol (FIG. 3B).

3-2(3). Islet Characteristics

The characteristics of the isolated islets are shown in Table 8 below.

TABLE 8 C1 Protocol C2 Protocol Kyoto Protocol Viability (%) 93 ± 4 96 ±1 96 ± 1 Morphological  6.9 ± 0.6  7.8 ± 0.6  8.8 ± 0.6* score Purity(%) 93 ± 1 86 ± 5 78 ± 9 Efficiency of 51 ± 5 38 ± 6  64 ± 6**purification (%) Pre- 146 ± 16 152 ± 16 176 ± 12 Purification Islet Size(μm) Post- 113 ± 33  83 ± 11   211 ± 55*** Purification Islet Size (μm)Stimulation  1.4 ± 0.9  1.4 ± 0.4  1.6 ± 0.3 Index The * in the tableindicates that the Kyoto protocol gave a substantially highermorphological score than the C1 protocol (P < 0.03). The ** in the tableindicates that the Kyoto protocol gave a substantially higher efficiencyof purification than the C1 protocol (P < 0.05) and the C2 protocol (P <0.01). The *** in the table indicates that the Kyoto protocol gave asubstantially larger post-purification islet size than the C2 protocol(P < 0.04).

As shown in Table 8, there was no significant difference in stimulationindex, viability, purity, or pre-purification islet size among the threegroups.

However, it was found that the results with the Kyoto protocol for themorphological score, efficiency of purification, and post-purificationislet size were particularly good.

3-2(4). Purification Speed

The purification speeds in the individual isolation protocols wereinvestigated. As a result, it was found that the purification solutioncould be delivered into the COBE 2991 cell processor at a rate of 60ml/min in the Kyoto protocol.

Against this, the purification solution delivery rate in the C1 and C2protocols, which used Ficoll solution as the purification solution, was10 to 20 ml/min.

These results showed that the rate of islet purification could be speedup by 3- to 6-fold using iodixanol as the density gradient reagent. Theprimary factor for this result was thought to be the low viscosity ofthe purification solution when iodixanol was used.

Example 4 Investigation of Protease Inhibitor

The following experiment was carried out in order to examine the type ofprotease inhibitor.

Islet yield was assessed using the same procedure as described above forthe Kyoto protocol, with the exception that the solutions described inTable 9 were used for the protection solution injected into the mainpancreatic duct and the preservation solution in the two-layer method.

TABLE 9 Protection Solution and Preservation Solution Islet Yield (IE/g)ET-Kyoto Solution 2103 ET-Kyoto + AEBSF Solution 3448 M-Kyoto Solution7253 (ET-Kyoto + Ulinastatin Solution) ET-Kyoto + Gabexate Mesilate 7140Solution

These results demonstrate that islet yield was increased when a proteaseinhibitor was added to the protection solution injected into thepancreatic duct.

It was found in particular that islet yield was substantially raisedwhen the protease inhibitor was ulinastatin or gabexate mesilate.

In addition, the use of ET-Kyoto+gabexate mesilate solution as theprotection solution injected into the main pancreatic duct and as thepreservation solution in the two-layer method yielded a purity of 89%and a viability of 90% for the isolated islets.

Example 5 Investigation of Purification

Further investigations were carried out using different purificationsolutions as follows.

The efficiency of purification was assessed by the method describedabove using the Kyoto protocol described in the above porcine isletisolation procedure, with the exception that the purification solutionsshown in Table 10 were used.

TABLE 10 Purification Solution Efficiency of purification (%) I-KyotoSolution 64 I-Kyoto + AEBSF Solution 70 MI-Kyoto Solution (I-Kyoto + 80ulinastatin solution)

As is shown in Table 10, the efficiency of purification was found to beincreased when a protease-inhibitor-containing solution was used as thepurification solution.

In particular, the use of MI-Kyoto solution, that is, the use ofulinastatin as the protease inhibitor, was found to provide asubstantial increase in efficiency of purification.

Further, just as for the use of I-Kyoto solution, the use of MI-Kyotosolution also enabled the purification solution to be delivered into theCOBE 2991 cell processor at a rate of 60 ml/min.

Example 6 Human Islet Isolation 6-1. Procedure for Human Islet Isolation

Human islet isolation was carried out using the same procedure as forporcine islet isolation, except for the points noted below.

With informed consent in place, thirteen human pancreases were acquiredfrom non-heart-beating donors through the Central Japan Region and theWestern Japan Region of the Japan Organ Transplant Network.

A catheter was inserted in order to rapidly cool the pancreases via theblood vessels of the donor's inguinal region and cold lactated Ringer'ssolution was injected through this catheter and circulated from afterheart beat cessation until removal of the pancreas.

The pancreases were then removed and a catheter was immediately insertedand M-Kyoto solution was injected into the main pancreatic duct at therate of 1 ml per 1 gram pancreas weight. This was quickly followed byimmersion of the pancreases in the preservation solution of a two-layermethod preservation container. The pancreases were then transported tothe GMP-grade Center for Cell and Molecular Therapy at Kyoto University.The two-layer method used M-Kyoto solution and liquid perfluorocarbon(PFC).

The assessment procedures were the same as in the previously describedporcine islet isolation procedure. Statistical evaluation was also thesame as for porcine islet isolation.

6-2. Isolation Protocol

Isolation of human islets was carried out by the C2 protocol in 2 of thethirteen cases and was carried out by the Kyoto protocol in 11 of thethirteen cases.

6-3. Characteristics of Human Donors and Pancreases

The mean donor age was 44±4 years. The period of ICU stay was 11±3 days.The BMI was 21±1 kg/m². The pancreas size was 87±6 g. Abnormalities wereobserved in the average blood chemistry values of all the donors.

The warm ischemic time was 7±3 minutes. The cold ischemic time was256±18 minutes. The warm ischemic time for all the pancreases wasminimized by the immediate circulation with cold lactated Ringer'ssolution. In addition, the cold ischemic time was shorter than 6 hoursin all cases.

There were no significant differences between the two protocols withregard to donor conditions, warm ischemic time, and cold ischemic time.

6-4. Assessment of Isolation Protocols

The results of the assessment of human islet isolation are shown inTable 11 for the two protocols (C2 protocol, Kyoto protocol). The isletyield (IE) indicates the total islet yield.

TABLE 11 Kyoto Protocol (N = C2 Protocol (N = 2) 11) Pre-Purification733,620 ± 249,440 526,657 ± 67,695 Islet Yield (IE) Post-Purification339,480 ± 14,905  410,376 ± 42,412 Islet Yield (IE) Efficiency of 51 ±15 81 ± 5 purification (%) Purity (%) 50 ± 10 51 ± 6 Viability (%) 94 ±6  97 ± 1 Morphological score 9.0 ± 1.0  9.8 ± 0.1 Tissue Volume (mL)8.0 ± 1.0  6.4 ± 0.8 Negative Gram Stain 2/2 11/11 Endotoxin (EU) 14.4 ±11.0  8.7 ± 3.6 Transplant Criteria 2/2 11/11 Qualification Transplanted1/2 10/11

There were no significant differences between the two protocols in thepre-purification islet yield or post-purification islet yield. The sameresults are also obtained in the purity, viability, and morphologicalscore between the two protocols.

The efficiency of purification was 1.6-times higher in the Kyotoprotocol than in the C2 protocol. The endotoxin level was lower for theKyoto protocol than for the C2 protocol.

With regard to the purification speed, the purification solution couldbe delivered into the COBE 2991 cell processor at the rate of 60 ml/minin the Kyoto protocol. This rate was 10 to 20 ml/min for the C2protocol.

Based on these results, it was confirmed that the use of I-Kyotosolution as the purification solution also provides a 3- to 6-timesfaster islet purification speed in human islet isolation.

The following tests were also carried out with the purificationsolution.

The efficiency of purification and islet yield were assessed using thesame procedures as the Kyoto protocol in the above-described human isletisolation, except that the purification solutions shown in Table 12 wereused.

TABLE 12 Efficiency of Total Islet Yield Purification Solutionpurification (%) (IE) I-Kyoto Solution 78 479409 MI-Kyoto Solution 84.2544535

As shown in Table 12, it was found that the efficiency of purificationwas also raised in human islet isolation when aprotease-inhibitor-containing solution was used as the purificationsolution. It was additionally found that the islet yield was alsoraised.

6-5. Transplant Qualifying Criteria

The following transplant qualifying criteria were established based onthe Edmonton protocol.

Islet Yield≧5,000 IE/kg (patient body weight)

Purity≧30%

Tissue volume≦10 mL

Viability≧70%

Endotoxin≦5 IE/kg (patient body weight)

Negative Gram stain

With regard to these criteria, all thirteen cases satisfied thetransplant qualifying criteria, with the exception of islet yield.

6-6. Islet Transplantation into Type-I Diabetic Patients

Of the thirteen cases described above, eleven cases, that is, one caseof those islets isolated according to the C2 protocol and ten cases ofthose islets isolated according to the Kyoto protocol were transplantedinto six type-I diabetic patients. The islets isolated in the remainingtwo cases were cryopreserved.

Four of the six patients received multiple-donor islet transplants. Twopatients received single-donor islet transplants.

The assessments made after islet transplantation were carried out usingthe following methods.

The serum blood glucose, insulin requirement, and hemoglobin A1c (HbA1c)were assessed daily before and after transplantation. The glucagonstimulation test was carried out before transplantation, on the 30^(th)and 60^(th) day after the first transplantation, and on the 30^(th) dayand 60^(th) day after the second transplantation. In the glucagonstimulation test, blood was taken for C-peptide measurement immediatelybefore the injection of 1 mg glucagon and 6 minutes after the injection.

6-7. Assessment After Islet Transplantation

After islet transplantation, none of the six patients suffered fromhypoglycemic unawareness and an improved blood glucose control could beobserved in all six. In addition, the start of insulin secretion wasconfirmed in all of the transplant cases based on C-peptide measurement.

While the mean insulin amount was 39.2±3.2 units at the time oftransplantation, it fell to 11.0±4.4 units (P<0.0005). Two patientsbecame insulin free; in two other patients the amount of insulindeclined to below 10 units; and in the two other patients the amount ofinsulin also declined.

The HbA1C level gradually declined in all six patients, and in all sixpatients the HbA1c level reached normal at about 3 months after islettransplantation, regardless of whether the transplantation was from asingle donor or multiple donors (FIG. 4A).

The mean HbA1c level of the six patients underwent substantialimprovement, improving from 7.5±0.4% at the time of transplantation to5.1±0.2% (P<0.0003).

Prior to transplantation, all the patients had undetectable C-peptidevalues (<0.1 ng/ml), while post-transplant the C-peptide values could bedetected. With regard to the C-peptide values after the firsttransplantation, the basal value was 0.29±0.06 ng/ml and thepost-stimulation value was 0.52±0.11 ng/ml (N=6) (FIG. 4B). Both thebasal C-peptide value and the post-stimulation C-peptide value weresubstantially improved after the second islet transplantation from thevalues for the first transplantation. After the second transplantation,the basal C-peptide value was 0.75±0.12 ng/ml (P<0.01) and thepost-stimulation value was 1.45±0.26 ng/ml (P<0.005) (N=3) (FIG. 4B).

6-8. Clinical Islet Transplantation

Islet transplantation was carried out in a clinical setting inaccordance with the Kyoto protocol described above. A secondtransplantation was carried out in the same manner on the same patientafter about 2 months.

The islet yield for the first transplantation was 354,384 IE and for thesecond transplantation was 474,234 IE.

The recipient was a 36-year-old female with a 22-year history of type Idiabetes; there had been frequent episodes of severe hypoglycemia andthere was a history of diabetic retinopathy.

Kidney function was normal and creatinine level was 0.7 mg/dl.Immunosuppressives were administered on day 0 and day 4post-transplantation in accordance with the Edmonton protocol, with theexception that 20 mg basiliximab was used instead of daclizumab.

To provide a pre-transplantation blood glucose level control, bloodglucose was measured before breakfast and before dinner at approximately2 month intervals for approximately the preceding 2 years.

Blood glucose prior to islet transplantation was very unstable andranged broadly from 20 mg/dl to 400 mg/dl.

After the first transplantation, however, blood glucose was maintainedin a narrow range and settled into a narrow range from 50 mg/dL to 150mg/dL (FIG. 5A).

The pre-transplantation insulin requirement was from 30 to 40 unit/day;however, the insulin intake gradually declined to 10 unit/day by 1 monthafter the first transplantation. Moreover, the patient was able tocompletely stop and achieve insulin free on day 20 after the secondtransplantation (FIG. 5B).

The transaminase value temporarily increased after the firsttransplantation, but did not reach or exceed 100 mg/dl and returned tonormal values within 3 weeks. The creatinine value remained at or below1.0 mg/dl, and blood urea nitrogen (BUN) stayed at normal values duringthe entire period of observation.

As is clear from the results given above, the present invention has beenshown to raise efficiency of purification and to raise the yield ofislets that satisfy the transplant qualifying criteria. In addition, thepresent invention has been shown to raise the purification speed and toenable islet isolation with good efficiency in a short period of time.

Moreover, the present invention has been shown to have the ability toacquire good amounts of good-quality islets from non-heart-beatingdonors. The present invention has also been shown to be highlysuccessful with regard to islet transplantation from humannon-heart-beating donors.

1-15. (canceled)
 16. A pancreatic islet isolation method comprising thesequential steps of: (a) injecting a protection solution containing aprotease inhibitor and approximately 4 to 50 mmol/L of potassium intothe pancreatic duct of a procured pancreas, wherein said proteaseinhibitor is at least one selected from the group consisting ofulinastatin, gabexate mesilate and nafamostat mesilate; (b) injecting acollagenase solution into said pancreas into which the protectionsolution has been injected and digesting the pancreas; and (c) purifyingthe digested pancreatic tissue using a purification solution containinga density gradient reagent wherein said density gradient reagent isFicoll solution or iodixanol.
 17. The isolation method according toclaim 16, wherein in (a) the protection solution is injected into thepancreatic duct in an amount of approximately 0.1 to 10 ml per 1 gramorgan weight.
 18. The isolation method according to claim 16, wherein in(c) the purification solution further contains trehalose.
 19. Theisolation method according to claim 16, wherein in (c) the densitygradient reagent is iodixanol.
 20. The isolation method according toclaim 16, wherein in (c) the purification solution further contains aprotease inhibitor.
 21. The method according to claim 16, wherein in (a)said protease inhibitor is ulinastatin.
 22. The method according toclaim 16, wherein in (a) said protease inhibitor is gabexate mesilate.23. The method according to claim 16, wherein in (a) said proteaseinhibitor is nafamostat mesilate.